The Preliminary Study On The Impression Of Mini-P-glycoprotein And P-glycoprotein In Rat Pancreatic Beta Cells

Objective To detect the existence of mini-P-glycoprotein in rat pancreatic beta cells and the impact of the Berberine, Cyclosporine A and Doxorubicin to the MDR1/abcbl gene expression.Methods1. Northern blotting was applied to analyze the expression of abcblb gene at mRNA level(1) Rat islets were isolated from pancreas and insulinoma cell lines (INS-1) were cultured, then the total RNAs of them were extracted. The detected cDNA were generated by RT-PCR with specific primers selected from the abcblb mRNA. The cDNA were purified and labeled by DIG-dUTP as the probe for northern blotting.(2) The RNAs of rat islets and INS-1cells were separated by formaldehyde modified electrophoresis, transferred to nylon membrane by siphon imprinting method and baked at120℃for30min.(3) The hybridization was carried out between probes and RNAs on the nylon membrane.(4) The hybridization results were detected by DIG Luminescent Detection Kit.2. Western blotting was used to analyze abcblb at protein expression level(1) Proteins from INS-1cells were extracted by RIPA lysate buffer and the test group contained "Protease Inhibitor Cocktail Tablets" but the compared group not.(2) The protein concentrations were determined by BCA protein assay kit and the proteins were applied to SDS-PAGE then transferred to PVDF membranes, blocked and washed. The membranes were incubated with C219(1:20) overnight at4℃and incubated with the second antibody after TBST washes. The bands on the membranes were visualized with HRP chemoluminescencereagents in dark room on X-ray films.3. Real-Time PCR were used to detect the impact of Berberine, Cyclosporine A and Doxorubicin to the impression of the abcblgene and apoptosis gene INS-1cells were treated by Berberine (0.2μM,1μM,5μM and20μM), Cyclosporine A (O.1μg/mL) and Doxorubicin (50ng/mL) for one week. The total RNAs of each group were extracted and cDNA were obtained by RT-PCR. Real-Time PCR were used to detect the impression of the MDRl/abcbl gene (abcbl, abcbla and abcblb) and apoptosis gene (Bcl-2and Caspase-3).Results The, hybridization of northern blotting indicated a single band. Two proteins of INS-1cell homogenate,170-kDa and65kDa, were recognized by the specific antibody to P-glycoprotein (C219). A170-kDa protein was predominant when the homogenate was protected by protease inhibitors, whereas only65-kDa protein was detected without the protease inhibitors protection. Real-Time PCR reflected the expression of abcbla-N was in high level in Berberine group (0.2μM,1μM and5μM)(P＜0.05).Conclusion (1) Combined with the results of the northern blotting and western blotting, it might be concluded that the65kDa mini-P-glycoprotein might be protein degradation.(2) Berberine may affect the expression of the abcbl relative genes but the affection of0.1μg/mL Cyclosporine A and50ng/mL Adriamycin on P-glycoprotein relative genes was not obvious.